Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the Sequence Listing. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which the invention pertains.
Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patients characteristically show prolonged bleeding times, borderline thrombocytopenia, and decreased von Willebrand factor (vWF) high molecular weight multimers and functional activity (1-5). PT-vWD appears to result from an abnormality of the platelet receptor for vWF, whereby patient platelets show an abnormally increased binding of circulating vWF. In the laboratory, this platelet hyperresponsiveness may be demonstrated with the use of low concentrations of ristocetin. Whereas normal platelets show little or no aggregation at ristocetin concentrations as low as 0.5 mg/ml, patient platelets typically show significant binding of vWF, together with strong aggregation, following stimulation by 0.5 mg/ml, or even lower, concentrations of ristocetin (1-3). The unique ability of desialylated vWF (asialo-vWF) to agglutinate patient platelets in the presence of the divalent-cation chelator EDTA has additionally been demonstrated (6). Platelets from patients with PT-vWD also show a characteristically increased binding of the monoclonal antibody C-34, which is directed against an epitope within the platelet glycoprotein (GP) Ib/IX complex (7). Although this complex is known to constitute the platelet's ristocetin-dependent receptor for vWF (8), identification of a unique structural abnormality within this complex that might underlie the functional abnormalities seen in PT-vWD had not yet been achieved.
The platelet GP Ib/IX receptor for vWF is believed to consist of a 1:1 heterodimeric complex (9) between GP Ib (160 kDa) and GP IX (17 kDa) in a noncovalent association. GP Ib in turn consists of a disulfide-linked 140-kDa .alpha. chain (GP Ib.alpha.) and 22-kDa .beta. chain (GP Ib.beta.) (10). A full-length cDNA for GP Ib.alpha. was isolated from human erythroleukemia (HEL) cells by Lopez et al. (11). Absolute identity of the HEL GP Ib.alpha. sequence with that obtained from the sequencing of nearly 800 nucleotides of cDNA obtained from human platelets was subsequently reported by Wicki et al. (12). Moreover, the human gene for GP Ib.alpha. has now been sequenced, and the entire coding region for the resulting protein has been shown to reside within a single exon (13,14).
Functional studies utilizing the water-soluble, extracellular portion of GP Ib.alpha. termed glycocalicin, and more particularly the 45-kDa amino-terminal region common both to glycocalicin and to the native GP Ib.alpha. molecule, strongly suggest that the actual binding of vWF occurs within this region (15-18). What roles the other constituents of the complex may play in the regulation of vWF binding to the receptor remain unknown.
Previous investigations of the substitution of single amino acids at critical positions in protein sequences have demonstrated significant changes in the three-dimensional structure of these proteins (39). For example, substitution of Val (or any non-cyclic L-amino acid) for Gly 12 in the ras-oncogeneencoded p21 protein results in local changes in the conformation of the protein that result in more global changes in the protein structure (39,40). This alteration in structure is associated with activation of the protein (39,40).
It is known that the linear sequence of amino acids in a protein determines its three-dimensional structure of conformation (41-44). This conformation is the one of lowest conformational energy (41-44). This principle allows computation of the structure of a polypeptide from its amino acid sequence. It is possible to compute the conformational energy for a particular conformation of a polypeptide using an equation which has been parameterized on a large body of experimental crystal structure and gas phase data and is contained in the computer program ECEPP (Empirical Conformational Energies of Peptides Program) (43). It is possible to generate the conformations that can be adopted by a given polypeptide chain. The conformational energies for each of these conformers is computed and then subjected to energy minimization (39,41). The resulting lowest energy conformer(s) is (are) then the one(s) that should be observed experimentally. The program ECEPP has been used to compute the low energy conformations for the single amino acid residues (45,46), oligopeptides (47), polypeptides such as gramicidin s and collagen (48,49), and proteins such as melittin (50), and avian pancreatic polypeptide, with excellent agreement between the predicted low energy structures and the corresponding experimentally determined ones (39,41,42).
It has been shown that the conformational preferences of an amino acid residue in a polypeptide chain are predominantly influenced by its four nearest neighbors on the amino and carboxyl terminal ends of this residue (51).